Human tissue plasminogen activator was first identified as a substantially pure isolate from a natural source, and tested for requisite plasminogen activator activity by Collen, et al. European Patent Application Publication No. 041766, published Dec. 16, 1981, based upon a first filing of Jun. 11, 1980.
Subsequently, researchers in Assignee's laboratories produced human tissue plasminogen activator, essentially free of proteins with which it is ordinarily associated, via recombinant DNA technology. This work has been recorded in the scientific literature and in European Patent Application Publication No. 093619 published Nov. 9, 1983, based upon a first filing on May 5, 1982. The above patent application (EPO Publication No. 093619) contemplates the production of various human plasminogen activator derivatives, variously modified by amino acid substitutions, deletions, additions, or replacements prepared, for example, by site directed mutagenesis of the underlying DNA.
As disclosed therein, human tissue plasminogen activator (t-PA) exists in both a single-chain and a two-chain form. The latter is a proteolytic derivative of the former. It has been shown that proteolytic conversion of the single-chain form to the two-chain form occurs during the lysis of a fibrin clot. Rijken, et al. J. Biol. Chem. 257, 2920 (1982). It is believed that the two-chain form is the agent responsible for plasminogen activator activity, although there have been some initial reports indicating that the single-chain form of human t-PA, Rijken, Ibid, and the single-chain form of porcine t-PA, Ranby, et al. Thromb. Res. 27, 176 (1982), may have some activity. See also Rijken et al., Biochim. Biophy. Acta 580 (1979).
Subsequent investigators, however, have dismissed such reports of single-chain activity as being the result of contamination of these preparations with low amounts of the two-chain form. Andreasen, et al. EMBO J. 3, 51 (1984); Ichinose, et al. FEBS. Letters 175, 412 (1984). Such subsequent reports have reaffirmed the general belief that serine proteases, including t-PA, are expressed as inactive, single-chain zymogens which only become active upon hydrolysis of the protein at a specific site, e.g., arginine at position 275 in the case of t-PA.
In vivo comparisons of the ability of one-chain verses two-chain plasminogen activator to lyse fibrin clots have been performed using rabbits and dogs. In rabbits, approximately equal potency has been observed for the two forms of this enzyme. Collen et al., J. Clin. Invest. 71, 368 (1983). When evaluated in a similar model in dogs, however, the one-chain form of plasminogen activator was reported to be slightly less active than the two-chain form. Korninger et al., J. Clin. Invest. 69, 573 (1982). These studies, therefore, indicate that one-chain plasminogen activator is no better than, and in fact may be less potent than, the two-chain form of plasminogen activator in their ability to dissolve fibrin clots in vivo.